Lower extremity Peripheral Vascular Disease (PAD) affects ~8-12 million adults in the US, and is a major cause of morbidity and mortality, with risk of cardiovascular mortality equivalent to that of coronary artery disease. Despite this, there are no specific medical treatments to improve blood flow to the limbs, and no diagnostic test for early detection or risk stratification of PAD. The tissue adaptive response to ischemia involves changes in gene expression in multiple pathways. Micro- RNAs (miRS) have been identified to be powerful regulators of gene expression, specifically in the context of tissue injury/ischemia. MiRs have great potential as therapeutic agents due to their ease of synthesis, stability, and ability of single miR to regulate several genes within a pathway. In addition, miRs are also found in the serum/plasma and are remarkably protected from RNAse degradation. As such, they have great potential for diagnostic and/or prognostic markers. The overall goal of this proposed research plan is to identify miRs with potential for use as biomarkers or therapeutic targets/agents in PAD, and to understand their mechanism(s) of actions(s). This proposal is designed to accomplish this goal using three complementary hypotheses-driven aims built on collaborations with established investigators. The first aim is designed to test the hypothesis tha cell-free miRs serve as paracrine factors in PAD. Using cell culture models, we will sequentially determine whether release of miRs from endothelial and muscle cells is a regulated, non-random process under different severities and durations of hypoxia. We will then test if this process is altered by established factors that modulate the extent of injury to ischemia, such as hyperglycemia. Lastly, we will test for paracrine effects of these cell free miRs on cellular function. The second aim is designed to test the hypothesis that circulating miRs in serum/plasma can be used as diagnostic/prognostic markers for PAD. This will be tested using existing serum samples from patients with varying severities of PAD, and selected miRs identified as clinically relevant will be checked for associations with the existing clinical outcomes database. Lastly, based on preliminary data, the third aim is designed to test the hypothesis that concomitant downregulation of antiangiogenic miR-222 and upregulation of pro-angiogenic miR-221 will improve limb perfusion and recovery from hind-limb ischemia in a mouse model of PAD. Together, the results from the proposed aims will help identify micro-RNAs that can be used as potential therapeutic tools or targets for patients with PAD. In addition, this will help identify circulating micro-RNAs that may have diagnostic/prognostic potential for patients with PAD.